Surface Of Lipid Droplets Research Articles

Quantitative Lipid Droplet Proteomics Reveals Mycobacterium tuberculosis Induced Alterations in Macrophage Response to Infection.

Growing evidence suggests the importance of lipid metabolism in pathogenesis of tuberculosis. Neutral lipids form the majority of lipids in a caseous granuloma, a pathology characteristic of tuberculosis. Cytosolic lipid droplets (LDs) of macrophages form the store house of these lipids and have been demonstrated to contribute to the inflammatory response to infection. The proteome of lipid droplets reflects the mechanisms of lipid metabolism active under a condition. However, infection induced changes in the proteome of these dynamic organelles remains elusive. Here, we employed quantitative proteomics to identify alterations induced upon infection with live Mycobacterium tuberculosis (Mtb) in comparison with heat killed bacilli or uninfected macrophages. We found increased abundance of proteins coupled with lipid metabolism, protein synthesis, and vesicular transport function in LDs upon infection with live Mtb. Using biochemical methods and microscopy, we validated ADP-ribosyltransferase (Arf)-like 8 (ARL8B) to be increased on the lipid droplet surface of live Mtb infected macrophages and that ARL8B is a bonafide LD protein. This study provides the first proteomic evidence that the dynamic responses to infection also encompass changes at the level of LDs. This information will be important in understanding how Mtb manipulates lipid metabolism and defense mechanisms of the host macrophage.

Syntaxin 17 Recruits ACSL3 to Lipid Microdomains in Lipid Droplet Biogenesis

During lipid droplet (LD) formation, several key enzymes for neutral lipid biosynthesis, such as acyl-CoA synthetase 3 (ACSL3), translocate from the bilayer of the endoplasmic reticulum membrane or mitochondria-associated membrane to the monolayer surface of LDs. It has been recently shown that syntaxin 17 (Stx17) in cooperation with synaptosomal-associated protein of 23 kDa (SNAP23) facilitates the translocation of ACSL3 from the endoplasmic reticulum/mitochondria-associated membrane to LDs. In this study, we investigated whether lipid microdomains enriched in cholesterol and sphingolipids are important for the formation of LDs and the interaction of Stx17 with ACSL3 and SNAP23. Cholesterol depletion and blockage of ceramide synthesis by chemicals inhibited oleic acid (OA)-induced LD biogenesis and decreased the interaction of Stx17 with ACSL3 and SNAP23, whereas blockage of ganglioside GD3 synthesis by sialyltransferase knockdown interfered with LD biogenesis by affecting the interaction of Stx17 with SNAP23 but not ACSL3. Consistent with the requirement of GD3 in LD biogenesis, Stx17 was found to associate with GD3-containing membranes upon OA loading. SNAP23 and a minor fraction of Stx17 were found to reside in detergent-resistant membranes (DRMs), whereas OA treatment caused redistribution of ACSL3 and Stx17 to DRMs. Importantly, the redistribution of ACSL3 to DRMs was abrogated upon depletion of Stx17 or SNAP23. Taken together, our results highlight the importance of lipid microdomains enriched in cholesterol and sphingolipids as a platform for the interaction of Stx17 with ACSL3 and SNAP23 in LD biogenesis.

Impairment of lipophagy by PNPLA1 mutations causes lipid droplet accumulation in primary fibroblasts of Autosomal Recessive Congenital Ichthyosis patients

BackgroundAutosomal Recessive Congenital Ichthyosis (ARCI) is a group of epidermal keratinization disorders. One of the disease-associated proteins, patatin-like phospholipase domain-containing protein-1 (PNPLA1), plays a key role in the epidermal omega-O-acylceramide synthesis and localizes on the surface of lipid droplets (LDs). ObjectivePreviously, routine clinical test results showed abnormal LD accumulation in blood smear samples of our ARCI patients with PNPLA1 mutations. To investigate the abnormal accumulation of LDs, we analyzed primary fibroblast cells of ARCI patients with PNPLA1 mutations (p.Y245del and p.D172N). We hypothesized that PNPLA1 mutations might affect lipophagy-mediated regulation of LDs and cause intracellular lipid accumulation in ARCI patients. MethodsLD accumulation was analyzed by fluorescence staining with BODIPY®493/503 in the fibroblasts of patient cells and PNPLA1 siRNA transfected control fibroblast cells. The expression of PNPLA1 and its effects on the lipophagy-mediated degradation of LDs were analyzed by immunocytochemistry and immunoblotting. ResultsOur results showed that mutant or downregulated PNPLA1 protein causes abnormal intracellular LD accumulation. We found that PNPLA1 mutations affect neither the cellular localization nor the expression levels of the protein in fibroblast cells. When we analyzed lipophagic degradation process, LC3 expression and the number of autophagosomes were significantly decreased in fibroblast cells of the patients. In addition, co-localization of LDs with autophagosomes and lysosomes were markedly less than that of the control group. ConclusionPNPLA1 mutations caused disturbances in both autophagosome formation and fusion of autophagosomes with lysosomes. Our results indicate a possible role for PNPLA1 protein in LD regulation via lipophagy-mediated degradation.

The GTPase ARFRP1 affects lipid droplet protein composition and triglyceride release from intracellular storage of intestinal Caco-2 cells

Intestinal release of dietary triglycerides via chylomicrons is the major contributor to elevated postprandial triglyceride levels. Dietary lipids can be transiently stored in cytosolic lipid droplets (LDs) located in intestinal enterocytes for later release. ADP ribosylation factor-related protein 1 (ARFRP1) participates in processes of LD growth in adipocytes and in lipidation of lipoproteins in liver and intestine. This study aims to explore the impact of ARFRP1 on LD organization and its interplay with chylomicron-mediated triglyceride release in intestinal-like Caco-2 cells. Suppression of Arfrp1 reduced release of intracellularly derived triglycerides (0.69-fold) and increased the abundance of transitional endoplasmic reticulum ATPase TERA/VCP, fatty acid synthase-associated factor 2 (FAF2) and perilipin 2 (Plin2) at the LD surface. Furthermore, TERA/VCP and FAF2 co-occurred more frequently with ATGL at LDs, suggesting a reduced adipocyte triglyceride lipase (ATGL)-mediated lipolysis. Accordingly, inhibition of lipolysis reduced lipid release from intracellular storage pools by the same magnitude as Arfrp1 depletion. Thus, the lack of Arfrp1 increases the abundance of lipolysis-modulating enzymes TERA/VCP, FAF2 and Plin2 at LDs, which might decrease lipolysis and reduce availability of fatty acids for triglyceride synthesis and their release via chylomicrons.

FAM134B promotes adipogenesis by increasing vesicular activity in porcine and 3T3-L1 adipocytes.

Family with sequence similarity 134, Member B (FAM134B), is a cis-Golgi transmembrane protein that is known to be necessary for the long-term survival of nociceptive and autonomic ganglion neurons. Recent work has shown that FAM134B plays a pivotal role in autophagy-mediated turnover of endoplasmic reticulum (ER) membranes, tumor inhibition and lipid homeostasis. In this study, we provide mechanistic links between FAM134B and ARF-related protein 1 (ARFRP1) and further show that FAM134B resides in the Golgi apparatus. Here, we found that FAM134B increased lipid accumulation in adipocytes. Transport vehicle number and ADP-ribosylation factor (ARF) family gene expression were also increased by FAM134B overexpression, suggesting that vesicular transport activity enhanced lipid accumulation. ARF-related protein 1 (ARFRP1) is a GTPase that promotes protein trafficking. We show that FAM134B regulates the expression of ARFRP1, and the knockdown of ARFRP1 abolishes enhancement on lipid accumulation caused by FAM134B. In addition, FAM134B upregulates the PAT family protein (PAT), which associates with the lipid droplets (LDs) surface and promotes lipolysis by recruiting adipocyte triglyceride lipase (ATGL). These findings indicate that FAM134B promotes lipid accumulation and adipogenic differentiation by increasing vesicle transport activity in the Golgi apparatus and inhibiting the lipolysis of LDs.

Coordination Among Lipid Droplets, Peroxisomes, and Mitochondria Regulates Energy Expenditure Through the CIDE-ATGL-PPARα Pathway in Adipocytes.

Metabolic homeostasis is maintained by an interplay among tissues, organs, intracellular organelles, and molecules. Cidea and Cidec are lipid droplet (LD)-associated proteins that promote lipid storage in brown adipose tissue (BAT) and white adipose tissue (WAT). Using ob/ob/Cidea-/- , ob/ob/Cidec-/- , and ob/ob/Cidea-/-/Cidec-/- mouse models and CIDE-deficient cells, we studied metabolic regulation during severe obesity to identify ways to maintain metabolic homeostasis and promote antiobesity effects. The phenotype of ob/ob/Cidea-/- mice was similar to that of ob/ob mice in terms of serum parameters, adipose tissues, lipid storage, and gene expression. Typical lipodystrophy accompanied by insulin resistance occurred in ob/ob/Cidec-/- mice, with ectopic storage of lipids in the BAT and liver. Interestingly, double deficiency of Cidea and Cidec activated both WAT and BAT to consume more energy and to increase insulin sensitivity compared with their behavior in the other three mouse models. Increased lipolysis, which occurred on the LD surfaces and released fatty acids, led to activated β-oxidation and oxidative phosphorylation in peroxisomes and mitochondria in CIDE-deficient adipocytes. The coordination among LDs, peroxisomes, and mitochondria was regulated by adipocyte triglyceride lipase (ATGL)-peroxisome proliferator-activated receptor α (PPARα). Double deficiency of Cidea and Cidec activated energy consumption in both WAT and BAT, which provided new insights into therapeutic approaches for obesity and diabetes.

Reprograming of Human Adipocytes to a Briter Phenotype—Enhanced Fatty Acid Oxidation and Lipid Droplet Remodeling

Converting energy storing white adipocytes to energy wasting ‘brite’ ones is now recognized to be feasible and may confer protection against obesity and its related cardiometabolic diseases. Fragments of omental and subcutaneous human adipose tissues were cultured for 7 days with insulin and dexamethasone with or without rosiglitazone (rosi) to induce britening. Rosi caused a metabolic reprograming of the adipocytes including increased rates of basal FA oxidation and the expression of mRNAs and proteins in multiple pathways that modulate FA metabolism, triacylglycerol synthesis, cellular FA trafficking, and mitochondrial oxidative capacity (PGC1a, UCP1, CIDEA, PLIN5, FABP3). Furthermore, these changes were associated with a dramatic remodeling of the surface of the adipocyte lipid droplet (LD) such that clusters of small LDs decorated with mitochondria were formed. Formation of these small LDs was blocked by inhibition of lipolysis and required FA activation and reesterification. Surprisingly, both omental and subcutaneous adipocytes were similarly ‘britened’. A similar remodeling was induced in primary cultures of newly-differentiated human adipocytes and these cells showed higher rates of basal and maximally-stimulated oxygen consumption. Consistent with transcriptome changes in both systems, lipidomics showed increased levels of unsaturated FAs, phospholipid species, and cardiolipins in brite cells. Taken together, these results identify a coordinated metabolic and structural reprogramming of mature human adipocytes toward a more oxidative phenotype. Although rosi has unacceptable clinical side effects, knowledge of the downstream effectors of this metabolic improvement may lead to development of targeted approaches to improve adipocyte and metabolic health and prevent type 2 diabetes in human obesity. Disclosure M. Lee: None. S. Jash: None. V. Puri: None. S.K. Fried: None.

Exendin-4 improves ER stress-induced lipid accumulation and regulates lipin-1 signaling in HepG2 cells.

Lipin-1 performs dual function during lipid metabolism, i.e., it functions as a transcriptional coactivator and as a phosphatidate phosphatase during triglyceride biosynthesis. We investigated whether exendin-4 prevented endoplasmic reticulum (ER) stress-induced hepatic steatosis and whether the protective effects of exendin-4 were associated with lipin-1 signaling. Tunicamycin and thapsigargin, ER stress inducers, increased triglycerides (TG) content and expression of genes encoding lipid droplet surface proteins. Exendin-4 decreased the expression of ER stress markers phosphorylated PKR like ER kinase (PERK), phosphorylated inositol-requiring enzyme 1 alpha (IRE1α), and glucose-regulated protein 78kDa (GRP78) proteins and spliced X-box binding protein 1 (XBP-1s) mRNA and increased the expression of genes encoding lipolytic enzymes hormone-sensitive lipase (HSL) and monoacylglycerol lipase (MGL) and VLDL assembly-associated proteins microsomal triglyceride transfer protein (MTP) and apolipoprotein B (APOB) in tunicamycin-pretreated cells. Moreover, exendin-4 significantly decreased lipin-1β/α ratio by increasing SFRP10 and increased lipin-1 nuclear localization. The decrease in lipin-1β/α ratio was also observed in SIRT1 and AMPK agonist-treated cells. These data suggest that exendin-4 improves ER stress-induced hepatic lipid accumulation by increasing lipolysis and VLDL assembly, which is partially mediated by the regulation of lipin-1 signaling.

Diacylglycerol acyltransferase-2 contains a c-terminal sequence that interacts with lipid droplets

Diacylglycerol acyltranferase-2 (DGAT2) is a resident protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerol. When lipid droplet formation is stimulated by incubating cells with fatty acids, DGAT2 becomes concentrated around the surface of cytosolic lipid droplets. Using confocal microscopy and directed mutagenesis, we have identified a 17-amino acid sequence in the C-terminal region of DGAT2 that is necessary and sufficient for targeting DGAT2 to lipid droplets. When this region was deleted, DGAT2 remained in the ER and did not target to lipid droplets. Fusing this sequence to mCherry directed the fluorescent reporter to lipid droplets. Similarly, when the corresponding region of monoacylglycerol acyltransferase-2 (MGAT2) was replaced with this sequence, MGAT2 was also targeted to lipid droplets. Lastly, we demonstrated that DGAT2 in ER membranes is continuous with lipid droplets. We propose a new model whereby DGAT2 remains in the ER during lipid droplet formation via it's transmembrane domains and interacts with nascent lipid droplets via its C-terminal lipid droplet interacting domain as they expand.

Reducing Intestinal Digestion and Absorption of Fat Using a Nature-Derived Biopolymer: Interference of Triglyceride Hydrolysis by Nanocellulose.

Engineered nanomaterials are increasingly added to foods to improve quality, safety, or nutrition. Here we report the ability of ingested nanocellulose (NC) materials to reduce digestion and absorption of ingested fat. In the small intestinal phase of an acellular simulated gastrointestinal tract, the hydrolysis of free fatty acids (FFA) from triglycerides (TG) in a high-fat food model was reduced by 48.4% when NC was added at 0.75% w/w to the food, as quantified by pH stat titration, and by 40.1% as assessed by fluorometric FFA assay. Furthermore, translocation of TG and FFA across an in vitro cellular model of the intestinal epithelium was significantly reduced by the presence of 0.75% w/w NC in the food (TG by 52% and FFA by 32%). Finally, in in vivo experiments, the postprandial rise in serum TG 1 h after gavage with the high fat food model was reduced by 36% when 1.0% w/w NC was administered with the food. Scanning electron microscopy and molecular dynamics studies suggest two primary mechanisms for this effect: (1) coalescence of fat droplets on fibrillar NC (CNF) fibers, resulting in a reduction of available surface area for lipase binding and (2) sequestration of bile salts, causing impaired interfacial displacement of proteins at the lipid droplet surface and impaired solubilization of lipid digestion products. Together these findings suggest a potential use for NC, as a food additive or supplement, to reduce absorption of ingested fat and thereby assist in weight loss and the management of obesity.

Encapsulation of vitamin D3 in pickering emulsions stabilized by nanofibrillated mangosteen cellulose: Impact on in vitro digestion and bioaccessibility

Oil-in-water Pickering emulsions stabilized by nanofibrillated cellulose (NFC) were used to encapsulate and deliver vitamin D3. NFC was extracted from a waste product of the food industry, mangosteen (Garcinia mangostana L.) rind, using dissolution in a hot sodium hydroxide solution, bleaching using hydrogen peroxide, and shearing using a high-pressure homogenizer. This yielded cellulose fibers with a diameter of around 60 nm and a length of several micrometers. Emulsions containing 10% w/w oil (0.01% w/w vitamin D3 and 9.99% w/w soybean oil), 0.10–0.70% w/w NFC as emulsifier, and phosphate buffer (pH 7) were prepared. The effect of NFC on lipid digestion and vitamin bioaccessibility was investigated using a simulated gastrointestinal tract (GIT) model, which included mouth, stomach and small intestine phases. The rate and extent of lipid digestion, as well as the vitamin bioaccessibility, decreased with increasing NFC concentration. Numerous physicochemical phenomena may account for this effect, including the ability of NFC to: act as a physical barrier at the lipid droplet surfaces; to promote droplet flocculation in the gastric phase; and, to increase the viscosity of the aqueous phase. The slight decrease in vitamin D3 bioaccessibility at higher NFC levels, was probably due to the lower level of lipid digestion. Our results indicate that mangosteen fiber can be used to stabilize oil-in-water emulsions, and only has a minor effect on lipid digestion and vitamin bioaccessibility when used at relatively low levels. This information may be useful for the rational design of functional foods from natural waste-products, such as mangosteen rind.

Abstract 045: SR-BI and PCPE2 Modulate Lipid Trafficking in Adipocytes

Adipose tissue undergoes distinct structural remodeling in response to increases in fat mass, as in obesity. During this process, the extracellular matrix (ECM) plays a pivotal role with respect to both adipocyte receptor signaling and structural remodeling. One ECM protein, procollagen endopeptidase enhancer 2 (PCPE2), has been shown by our lab to work in partnership with the scavenger receptor class B type I (SR-BI) to regulate adipocyte cholesterol homeostasis. To investigate the functional impact of the partnership between SR-BI and PCPE2, we studied mature adipocytes differentiated from murine embryonic fibroblasts (MEF) and compared to our adipose specific PCPE2 knockout mice. Our studies showed the presence of an SR-BI-PCPE2 protein complex based on the co-immunoprecipitation of PCPE2 with SR-BI in both differentiated MEFs and mouse adipose tissue. Furthermore, stimulated emission depletion (STED) microscopy showed SR-BI and PCPE2 localized on the lipid droplet surface separated by distances ranging from 24 – 90 nm, suggesting that the SR-BI-PCPE2 complex may contain other proteins. Most significantly, a loss of SR-BI function was shown in PCPE2-deficient adipocytes by the SR-BI mediated HDL binding and cholesteryl ester (CE) uptake assays. Compared to differentiated MEFs from Ldlr -/- mice, binding of HDL to differentiated MEFs from Ldlr -/- Pcpe2 -/- mice was reduced by 75%, and HDL cholesteryl oleyl ether uptake was reduced by 50%. Disruption of adipocyte SR-BI function in the absence of PCPE2 also reduced free cholesterol efflux indicating substantial disruption to intracellular cholesterol homeostasis. Moreover, analysis of RNAseq from several human clinical studies show that PCPE2 mRNA expression is positively correlated with fat mass in specific adipose depots, while negatively correlated with plasma insulin, glucose and triglyceride levels, with the latter two more robust in type 2 diabetic populations (FDR < 0.004). Overall, these results suggest that the partnership between SR-BI and PCPE2 in adipocytes plays an important role in cholesterol trafficking and homeostasis which influences fat mass expansion, glucose tolerance and insulin resistance.

Impact of Interfacial Composition on Lipid and Protein Co-Oxidation in Oil-in-Water Emulsions Containing Mixed Emulisifers.

The impact of interfacial composition on lipid and protein co-oxidation in oil-in-water emulsions containing a mixture of proteins and surfactants was investigated. The emulsions consisted of 5% v/v walnut oil, 0.5% w/v whey protein isolate (WPI), and 0 to 0.4% w/v Tween 20 (pH 3 and pH 7). The protein surface load, magnitude of the ξ-potential, and mean particle diameter of the emulsions decreased as the Tween 20 concentration was increased, indicating the whey proteins were displaced by this nonionic surfactant. The whey proteins were displaced from the lipid droplet surfaces more readily at pH 3 than at pH 7, which may have been due to differences in the conformation or interactions of the proteins at the droplet surfaces at different pH values. Emulsions stabilized by whey proteins alone had relatively low lipid oxidation rates when incubated in the dark at 45 °C for up to 8 days, as determined by measuring lipid hydroperoxides and 2-thiobarbituric acid-reactive substances (TBARS). Conversely, the whey proteins themselves were rapidly oxidized, as shown by carbonyl formation, intrinsic fluorescence, sulfhydryl group loss, and electrophoresis measurements. Displacement of whey proteins from the interface by Tween 20 reduced protein oxidation but promoted lipid oxidation. These results indicated that the adsorbed proteins were more prone to oxidation than the nonadsorbed proteins, and therefore, they could act as better antioxidants. Protein oxidation was faster, while lipid oxidation was slower at pH 3 than at pH 7, which was attributed to a higher antioxidant activity of whey proteins under acidic conditions. These results highlight the importance of interfacial composition and solution pH on the oxidative stability of emulsions containing mixed emulsifiers.

Hormone-sensitive lipase preferentially redistributes to lipid droplets associated with perilipin-5 in human skeletal muscle during moderate-intensity exercise.

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) are the key enzymes involved in intramuscular triglyceride (IMTG) lipolysis. In isolated rat skeletal muscle, HSL translocates to IMTG-containing lipid droplets (LDs) following electrical stimulation, but whether HSL translocation occurs in human skeletal muscle during moderate-intensity exercise is currently unknown. Perilipin-2 (PLIN2) and perilipin-5 (PLIN5) proteins have been implicated in regulating IMTG lipolysis by interacting with HSL and ATGL in cell culture and rat skeletal muscle studies. This study investigated the hypothesis that HSL (but not ATGL) redistributes to LDs during moderate-intensity exercise in human skeletal muscle, and whether the localisation of these lipases with LDs was affected by the presence of PLIN proteins on the LDs. HSL preferentially redistributed to PLIN5-associated LDs whereas ATGL distribution was not altered with exercise; this is the first study to illustrate the pivotal step of HSL redistribution to PLIN5-associated LDs following moderate-intensity exercise in human skeletal muscle. Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) control skeletal muscle lipolysis. ATGL is present on the surface of lipid droplets (LDs) containing intramuscular triglyceride (IMTG) in both the basal state and during exercise. HSL translocates to LD in ex vivo electrically stimulated rat skeletal muscle. Perilipin-2- and perilipin-5-associated lipid droplets (PLIN2+ and PLIN5+ LDs) are preferentially depleted during exercise in humans, indicating that these PLINs may control muscle lipolysis. We aimed to test the hypothesis that in human skeletal muscle in vivo HSL (but not ATGL) is redistributed to PLIN2+ and PLIN5+ LDs during moderate-intensity exercise. Muscle biopsies from 8 lean trained males (age 21±1years, BMI 22.6±1.2kgm-2 and V̇O2 peak 48.2±5.0mlmin-1 kg-1 ) were obtained before and immediately following 60min of cycling exercise at ∼59% V̇O2 peak . Cryosections were stained using antibodies targeting ATGL, HSL, PLIN2 and PLIN5. LDs were stained using BODIPY 493/503. Images were obtained using confocal immunofluorescence microscopy and object-based colocalisation analyses were performed. Following exercise, HSL colocalisation to LDs increased (P<0.05), and was significantly greater to PLIN5+ LDs (+53%) than to PLIN5- LDs (+34%) (P<0.05), while the increases in HSL colocalisation to PLIN2+ LDs (+16%) and PLIN2- LDs (+28%) were not significantly different. Following exercise, the fraction of LDs colocalised with ATGL (0.53±0.04) did not significantly change (P<0.05) and was not affected by PLIN association to the LDs. This study presents the first evidence of exercise-induced HSL redistribution to LDs in human skeletal muscle and identifies PLIN5 as a facilitator of this mechanism.

A giant amphipathic helix from a perilipin that is adapted for coating lipid droplets

How proteins are targeted to lipid droplets (LDs) and distinguish the LD surface from the surfaces of other organelles is poorly understood, but many contain predicted amphipathic helices (AHs) that are involved in targeting. We have focused on human perilipin 4 (Plin4), which contains an AH that is exceptional in terms of length and repetitiveness. Using model cellular systems, we show that AH length, hydrophobicity, and charge are important for AH targeting to LDs and that these properties can compensate for one another, albeit at a loss of targeting specificity. Using synthetic lipids, we show that purified Plin4 AH binds poorly to lipid bilayers but strongly interacts with pure triglycerides, acting as a coat and forming small oil droplets. Because Plin4 overexpression alleviates LD instability under conditions where their coverage by phospholipids is limiting, we propose that the Plin4 AH replaces the LD lipid monolayer, for example during LD growth.

Effects of phosphorylation state on perilipin 5 localization and trafficking.

Perilipins are a family of five proteins found on the surface of lipid storage droplets in nearly all tissues. These proteins act as cofactors for lipases and scaffolding for other proteins involved in lipid metabolism. In addition to the lipid droplet surface, members of the perilipin family have been found in the cytosol, endoplasmic reticulum, plasma membrane and mitochondria. The localization of these proteins is in part determined by their phosphorylation state. Recently perilipin 5 has been shown to localize to the nucleus when phosphorylated by PKA on serine 155 and interact with the peroxisome proliferator‐activated receptor gamma coactivator alpha (PGC‐1a). In silico analysis of a phosphomimetic mutant (Ser155Asp) indicate that this site may contain a nuclear localization sequence. Using a CHO model cell line we observed that translocation to the nucleus could be stimulated by treatment with forskolin and isobutylmethylxanthine (IBMX) and occurred independent of treating cells with oleic acid to promote triacylglycerol deposition. We are currently investigating the affects of stimulating other phosphorylation pathways to determine if they also phosphorylate perilipin 5 and affect subcellular localization. These data have the potential to gain insights to the regulation of perilipin 5 trafficking into the nucleus and therefore interacting with PGC‐1a regulating mitochondrial biogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipid Droplet Size Determines Cooperation of Lipolysis and Lipophagy in Hepatocytes.

Fatty liver disease affects 80–100 million Americans and is a precursor to irreversible and sometimes fatal liver damage. The initial stages of fatty liver disease are marked by the hepatocellular accumulation of fat‐storage organelles known as lipid droplets (LDs) that are degraded by two pathways: lipolysis that involves the recruitment of cytosolic lipases (ATGL, HSL) to the LD surface, and lipophagy that relies on autophagic membranes to target and traffic LDs for lysosomal degradation. Importantly, the mechanisms underlying a cooperation between these seemingly distinct pathways are not well understood. Previous studies have shown that the autophagic engulfment of cytosolic cargo is size‐restricted. Because of this, we HYPOTHESIZE that lipolysis and lipophagy occur as sequential processes initiated by cytosolic lipases to reduce LD size and allow for subsequent engulfment by autophagic membranes.MethodsIn AML12 hepatocytes, immunofluorescence microscopy and biochemical approaches were used to determine the enrichment of lipolysis vs lipophagy machinery on large vs. small LDs. Live‐cell microscopy was used for the first time to monitor LDs transitioning from lipolysis to lipophagic engulfment.ResultsConfocal microscopy of AML12 cells immunolabeled for adipose triglyceride lipase (ATGL) revealed a preferential association with large LDs with an average area of 4.0 μm2, seven‐fold larger than LDs not associated with ATGL. In striking comparison, LDs associated with components of the autophagic machinery (LC3) were two‐fold smaller in area (0.39 μm2). Consistent with this observation was the finding that siRNA knockdown of ATGL resulted in the persistence of large LDs (2.8 μm2), whereas knockdown of lysosomal acid lipase (LAL) left cells filled with many LDs of a smaller size (1.5 μm2). These observations were mimicked by inhibitors of ATGL (atglistatin) and lysosomal acidification (chloroquine). In support of these findings, differential centrifugation of AML12 homogenates allowed for separation of two distinct populations of LDs: a buoyant fraction of large LDs enriched in the cytosolic lipase ATGL, and a second collection of smaller, less buoyant LDs enriched in lysosomal enzymes such as Lamp2A and the autophagosome marker LC3‐II. Most interesting are time lapse movies of live cells showing engulfment of small LDs by a LAMP3‐positive late endosomal/lysosomal compartment, suggesting that these organelles play an important role in the direct targeting and engulfment of LDs for lipophagy.ConclusionHepatocytes appear to catabolize LDs in a tandem stepwise process that first utilizes cytosolic lipases to substantially reduce LD size, thereby allowing for subsequent engulfment by autophagosomes as well as late endosomes and lysosomes.Support or Funding InformationThis study was generously supported by funding from NIAAA R01AA020735, NIDDK T32DK007352, and the Mayo Clinic Robert and Arlene Kogod Center on Aging.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipid Droplet Membrane Protein and Monolayer Lipid Membranes Are Regulators of the Lipid Droplet Size: The Characterizations of Lipid Droplet Surface Decide the Size of Lipid Droplet

Lipid Droplet Membrane Protein and Monolayer Lipid Membranes Are Regulators of the Lipid Droplet Size: The Characterizations of Lipid Droplet Surface Decide the Size of Lipid Droplet

The metabolic capacity of lipid droplet localized acyl-CoA synthetase 3 is not sufficient to support local triglyceride synthesis independent of the endoplasmic reticulum in A431 cells

ACSL3 is the only long chain fatty acyl-CoA synthetase consistently found on growing and mature lipid droplets (LDs), suggesting that this specific localization has biological relevance. Current models for LD growth propose that triglycerides are synthesized by enzymes at the LD surface, with activated fatty acids provided by LD localized ACSL3, thus allowing growth independent of the ER. Here, we tested this hypothesis by quantifying ACSL3 on LDs from human A431 cells.RNAi of ACSL3 reduced the oleoyl-CoA synthetase activity by 83%, suggesting that ACSL3 is by far the dominant enzyme of A431 cells. Molar quantification revealed that there are 1.4 million ACSL3 molecules within a single cell. Metabolic labeling indicated that each ACSL3 molecule contributed a net gain of 3.1 oleoyl-CoA/s. 3D reconstruction of confocal images demonstrated that 530 individual lipid droplets were present in an average oleate fed A431 cell. A representative single lipid droplet with a diameter of 0.66 μm contained 680 ACSL3 molecules on the surface. Subcellular fractionation showed that at least 68% of ACSL3 remain at the ER even during extensive fatty acid supplementation. High resolution single molecule microscopy confirmed the abundance of cytoplasmic ACSL3 outside of LDs. Model calculations for triglyceride synthesis using only LD localized ACSL3 gave significant slower growth of LDs as observed experimentally.In conclusion, although ACSL3 is an abundant enzyme on A431 LDs, the metabolic capacity is not sufficient to account for LD growth solely by the local synthesis of triglycerides.

Specific Coating of Cellular Lipid Droplets by a Giant and Repetitive Amphipathic Helix

Lipid droplets (LDs) are dynamic organelles that play an essential role in cellular lipid homeostasis; however, the mechanisms of selective protein targeting to LDs to mediate their function are poorly understood. Many LD proteins interact with LDs via amphipathic helices (AHs), which can mediate direct and reversible binding to lipid surfaces. We use a uniquely long and monotonous AH, found in the mammalian LD protein perilipin 4, to probe the physico-chemical properties of the LD surface. We show that this AH is completely unstructured in solution but can adopt a highly helical conformation, over a length of 660 amino acids, when in contact with lipids. By the use of fluorescently-tagged AH variants, we show that LDs are relatively permissive for AH binding, suggesting a surface with abundant lipid packing defects, in agreement with predicted behavior of a phospholipid monolayer on a neutral lipid core (Bacle et al., 2017, Biophys J, 112:1417). Whereas a slight increase in the hydrophobicity of perilipin 4-derived AHs makes them more promiscuous for binding to other cellular compartments, perturbing the distribution of charged residues in the polar face causes a striking loss of LD targeting. These results suggest that the physico-chemistry of the perilipin 4 AH is exquisitely tuned to ensure LD specificity. In vitro, we find that the purified wild-type AH binds poorly to bilayer membranes. In contrast, it can interact efficiently with neutral lipids and is capable of forming small uniformly-coated oil droplets. Accordingly, overexpression of this AH in cells overcomes a decrease in LD stability associated with phospholipid depletion. We propose that by substituting for the phospholipid monolayer, perilipin 4 may be important for stabilization of LDs when phospholipids are limiting, for example during periods of LD growth.